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Use of High Throughput Assays and Computational Tools; Endocrine Disruptor Screening Program; Notice of Availability and Opportunity for Comment’’

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Use of High Throughput Assays and Computational Tools; Endocrine Disruptor Screening Program; Notice of Availability and Opportunity for Comment’’ NRDC EPA-HQ-OPPT-2015-0305 August, 2015 Comments from the Natural Resources Defense Council On The Document Titled, ‘‘Use of High Throughput Assays and Computational Tools; Endocrine Disruptor Screening Program; Notice of Availability and Opportunity for Comment’’ To the U.S. Environmental Protection Agency Docket No. EPA-HQ-OPPT-2015-0305 August 18, 2015 Background The Natural Resources Defense Council ("NRDC") is a national, non-profit environmental organization of lawyers, scientists, and other professionals. NRDC presents these comments on behalf of our 1.4 million members and online activists. NRDC does not have any financial interest in the topic of these comments. The endocrine system utilizes highly complex, tightly controlled molecular processes for its optimal functioning in the body. The proper balance of hormones can be synchronized in a variety of ways (including direct protein binding, epigenetic alterations, gene activation and silencing), and is essential across the entirety of the life course. Small changes in the perfectly orchestrated symphony of hormone levels can severely disrupt the harmony necessary for critical windows of development (e.g., fetal development, infanthood, childhood, and adolescence), leaving the body vulnerable to a host of negative health outcomes (such as diabetes, cancer, obesity, and reproductive dysfunction). The last decade has seen an exponential increase in the development of computational, biological, and chemical tools capable of increasing both the number of chemicals analyzed and the pace of chemical toxicity evaluation. These tools, including the EPA Toxicity Forecaster (ToxCast™) and the National Institute of Environmental Health Sciences (NIEHS) Tox21 platforms, have the potential to rapidly generate molecular and cellular data for thousands of chemicals at once, and provide an additional stream of useful information that can aide in regulatory decision-making. A program that evaluates chemicals for their ability to disrupt the endocrine system (also known as endocrine disrupting chemicals or EDCs) must be both protective and productive to sufficiently 1 NRDC EPA-HQ-OPPT-2015-0305 August, 2015 safeguard the health of the public. To be productive, the program must test and identify high priority EDCs as quickly as possible and prohibit them from entering or remaining in commerce. To be protective, the program must minimize the number of potentially harmful chemicals that are missed via testing and screening procedures (protect against false negatives). Created in response to the mandate in the 1996 Food Quality Protection Act, the Endocrine Disruptor Screening Program (EDSP) was established by the EPA to identify chemicals that could negatively impact the endocrine systems of humans and wildlife. In nearly 20 years since it was conceived of in the law, no chemical has been fully evaluated and regulated as a result of the EDSP, and thus the program has not been protective or productive. Unfortunately, EPA’s request for comments on the use of high throughput assays and computational tools in the Endocrine Disruptor Screening Program does not address the lack of protection and productivity of the program. In fact, the proposal could exacerbate the flaws – especially the lack of protectiveness in the EDSP - by relying on a model that produces an unacceptably high rate of false negatives, does not evaluate all known relevant biological pathways1, and has the potential to miss chemicals that could adversely impact large subsets (e.g., developing fetuses, infants, and children) of our population. In the comments presented below, we will outline both the deficiencies in the agency's proposed use of emerging technology, as well as offer possible alternative ways to more fully and effectively utilize emerging data streams to protect public health. Our comments will be presented in two parts. First, to be responsive to the agency request for comments on the use of the ToxCast™ “ER Model” as an alternative to the uterotrophic, ER binding, and ER transactivation (ERTA) EDSP Tier 1 assays, we will provide a technical critique of the “ER Model” and its uses. Second, we will provide recommendations for the use of emerging sources of data (including ToxCast™) to help increase the productivity and public health protective ability of the EDSP. NRDC does not support the use of the ToxCast™ “ER Model” as an alternative to the uterotrophic, ER binding, or ER transactivation Tier 1 assays. After extensive review of the scientific literature and background material provided with the FR notice, NRDC does not support the use of the “ER Model” as a replacement for the uterotrophic, ER binding, and ERTA EDSP Tier 1 assays2. We are very concerned with the inability of the “ER Model” to identify 1 Pathway-based approaches could be useful for evaluating the toxicity of chemicals that act via known molecular cascades, but can be unprotective when used to evaluate the toxicity of chemicals with unknown and/or multiple ways to disrupt a cellular process. For example, chemicals can act via different pathways depending upon the organ exposed (e.g., tamoxifen inhibits cell proliferation in breast cancer cells but can stimulate proliferation in the uterus). Large-scale efforts aimed at identifying the toxicity of large pools of chemically and physically distinct environmental chemicals should, therefore, not limit themselves by relying solely on overly simplified pathway- based models to identify and characterize chemical hazard and/or risk. 2 Though there are well-documented issues with the specificity and sensitivity of the uterotrophic, ER binding, and ERTA EDSP Tier 1 assays, these assays are part of a more robust battery of tests. Replacing three tests in a larger battery with a single approach (e.g., the “ER Model”) requires a high level of confidence (and low level of 2 NRDC EPA-HQ-OPPT-2015-0305 August, 2015 chemicals with well-established estrogenic activity (i.e., the model has too high a false negative rate), the limited statistical characterization and transparency of the model, and the overreliance on a narrowly focused pathway-based model that is populated with data from a convenience sample of high- throughput assays3. We are also troubled by the agency’s disregard of the FIFRA Scientific Advisory Panel (SAP) recommendations4 to forgo replacement of the uterotrophic assay with the “ER Model” without further testing. A. Limitations in the “ER Model” preclude it from replacing the EDSP Tier 1 uterotrophic assay. There are at least four limitations in the ToxCast™ “ER Model” that prohibit its use as a replacement for the in vivo uterotrophic assay. First, as noted by the FIFRA SAP in its review of the “ER Model,”5 the inability of in vitro assays to detect or account for altered chemical behavior due to biological processes such as metabolism and absorption severely limits the model’s ability to predict toxicity in whole animal systems. Chemical toxicity can change when a substance is bound to biomolecules or broken down by metabolism in the body. Cell-free and cell-based assays that lack metabolic capabilities, like those used in the ToxCast™ platform, could therefore miss toxicity that would otherwise occur in an intact living system (e.g., a human body). Second, the pathway modeled by EPA is merely a proxy for actual biological activity, and represents only one possible way in which estrogenic compounds can disrupt cellular processes. Chemicals can perturb estrogen signaling via genomic and non-genomic pathways, and can act as agonists (substances that turn on a signaling pathway) or antagonists (substances that turn off a signaling pathway). By evaluating only nuclear receptor-mediated (genomic) agonist behavior, the “ER Model” will miss chemicals that that interfere with normal estrogen signaling processes by other mechanisms, significantly reducing the usefulness of the model to identify estrogenic chemicals. Third, when compared to guideline and guideline-like studies, the “ER Model” demonstrated abysmal detection of estrogenic chemicals. The model missed nearly 30 percent (15/556) of the uncertainty) in the ability of the replacement approach to identify endocrine disrupting chemicals. The “ER Model” fails to meet this basic threshold. 3 The assays used in ToxCast™ were assembled from existing, “off-the-shelf” assays that could be purchased and/or resourced out (performed in contract labs) by EPA. The ToxCast™ assays were not designed to specifically test for activity on the proposed ER pathway. EPA’s approach is in stark contrast to that taken by NIEHS in their Tox21 program. As opposed to developing a “pathway-based model” to fit readily available assays, the Tox21 approach seeks to map biologically-relevant pathways first, then develop or modify assays to measure pathway-specific responses. By building a model to fit the available assays, the EPA approach is akin to forcing round pegs (biological mechanism) to fit into square holes (available assays). 4 FIFRA Scientific Advisory Panel (2015). Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held on “Integrated Endocrine Bioactivity and Exposure-Based Prioritization and Screening.” 5 Referred to as the “AUC Model” in footnote 4. 6 For this discussion, false negatives include chemicals that were incorrectly identified as not having estrogenic activity and those with “inconclusive” results. 3 NRDC EPA-HQ-OPPT-2015-0305 August, 2015 in vivo estrogenic reference chemicals7 from guideline-like studies and 3
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